Analysis of spermatogenesis in non-obstructive azoospermic and virtually azoospermic men with known testicular pathology.

It is widely thought that human testicles affected by unilateral pathology will have greater impairment of spermatogenesis than the otherwise unaffected testis. This study reviewed records of non-obstructive azoospermic (NOA) and virtually azoospermic (NOVA) men with associated testicular pathology who underwent testicular fine needle aspiration (FNA) mapping. Concentration of spermatozoa found in each testis was analysed to discern sperm-lateralization patterns in affected and unaffected testes. A total of 1098 FNA sites from 56 men (32 varicocele, 16 cryptorchidism, three epididymo-orchitis, two mumps orchitis, three torsion) were analysed. Overall, 38 patients (68%) had spermatozoa detected in at least one testis. Most men (68%) had equal proportions of FNA sites showing spermatozoa from both testes, 29% had more spermatozoa from the unaffected testis and 3% had more spermatozoa from the affected testis. Significantly fewer sperm-positive sites were observed on the affected (272 out of 752) than unaffected side (164 out of 346) (P < 0.0001, chi-squared test). When assessed by FNA mapping, most NOA and NOVA men with known unilateral testis pathology will have equal proportions of spermatozoa in both testes. However, when sperm production differs between sides, the unaffected side is much more likely to have spermatozoa. This information may be used to refine sperm-retrieval strategies in selected patients.

Immunofluorescent synaptonemal complex analysis in azoospermic men.

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.

Testicular fine-needle aspiration in infertile men: correlation of cytologic pattern with biopsy histology.

Open testicular biopsy is the standard method for histopathologic assessment of spermatogenesis. The need for testis biopsy has been questioned with the increased success of minimally invasive techniques such as fine-needle aspiration (FNA) mapping. This study examines whether FNA can provide cytologic information equivalent to histologic patterns by correlating diagnoses from testis FNA cytology with biopsy histology. Men (n = 87) who had undergone both diagnostic FNA mapping and open testis biopsy in the evaluation of infertility were identified. Biopsies were assessed by recognized histologic patterns of normal, hypospermatogenesis, early and late maturation arrest, and Sertoli cell only. FNA cytologic specimens were examined for adequacy and were classified similarly. Mixed patterns were also identified. The correlation between the two methods was 94%, with no differences among the different histologies. Discrepancies between cytology and histology were primarily the result of inadequate sampling and evidence of mixed patterns on FNA mapping. FNA cytology is a minimally invasive method of obtaining testicular tissue for diagnostic purposes. These data demonstrate that FNA cytology can evaluate accurately all classically defined histologic types, and may have the potential to replace testis biopsy in the assessment of spermatogenesis.

Role of uridine phosphorylase in the anabolism of 5-fluorouracil.

The activities of enzymes responsible for activating 5-fluorouracil (FUra) to 5-fluorouridine-5'-monophosphate (FUMP) were compared in normal and tumor tissues of rodents to assess the potential capacity of uridine phosphorylase to anabolize FUra to the nucleoside in the presence of ribose-1-phosphate (R-1-P). The activity of the alternative pathway to FUMP with a pyrimidine phosphoribosyltransferase [FUra + 1-pyrophosphoribosyl-5-phosphate (PPRP)] was approximately 15 to 17 nmoles/mg protein/hr in bone marrow from mice and rats and ranged from 28 to 47 nmoles/mg protein/hr in tumor tissues. Uridine phosphorylase [measured as the formation of 5-fluorouridine (FUrd) from FUra and R-1-P] was 35-230 nmoles/mg/hr in bone marrow and in two FUra-sensitive solid tumors, colon tumor No. 38 in mice and RPMI colon tumor in rats; the activity of uridine phosphorylase from L5178Y ascites leukemic cells was notably lower, 8 nmoles/mg/hr. Levels of uridine kinase ranged from 55 to 187 nmoles/mg protein/hr. Thus, the activities of the enzymes of the two-step FUra activating pathway were high compared to the PPRP-dependent activity in all tissues except L5178Y; also, the FUra-sensitive tumors yielded extracts with 1.5 to 6.5 times greater enzyme activity than the corresponding activity in bone marrow. Uridine phosphorylase was partially purified from rat liver, RPMI rat tumor and colon tumor No. 38; the apparent Km of FUra averaged 50 microM, almost 9-fold lower than that of uracil, and the apparent Km of R-1-P for condensation with FUra was 33 microM. The tissue concentration of R-1-P was greater than 70 microM in kidney and liver of rodents and somewhat less in spleen. Colon tumor No. 38 and RPMI colon tumor had 12 and 20 microM R-1-P, respectively, but these low values may reflect low tumor viability. The high levels of uridine phosphorylase and uridine kinase activities in normal tissues and even higher levels in tissues from FUra-sensitive tumors, as well as the sufficient concentration of R-1-P relative to its kinetic constant, suggest that FUra metabolism by the two-step pathway to FUMP may be a significant factor in the activity and selectivity of FUra.

Systematic fine-needle aspiration of the testis: correlation to biopsy and results of organ "mapping" for mature sperm in azoospermic men.

OBJECTIVES: This study compares fine-needle aspiration (FNA) and testis biopsy for the ability to detect mature sperm in the testes of azoospermic men. In addition, we introduce the concept of testis "mapping" with FNA and apply it to detect sperm in men with severe testis failure. METHODS: Sixteen patients were evaluated for azoospermia in a university-based infertility clinic. All men had testis biopsies and FNAs from matched testicular sites to assess for the presence of spermatozoa. Adequacy criteria for FNA specimens were strictly defined, and correlative analysis of the two techniques was performed. In addition, a subset of 12 men with nonobstructive azoospermia (NOA) had systematic FNA mapping (more than four FNA sites per testis) to detect mature sperm for potential clinical use. RESULTS: Adequate FNA specimens were obtained in 115 (91.3%) of 126 FNA attempts. Of 34 paired biopsy FNA sites. FNA was seen to be more sensitive than, and equally specific as, testis biopsy for sperm detection. When compared with the biopsy touch imprint, FNA was equally as sensitive and specific. Among men with NOA who underwent FNA mapping, 4 (33%) of 12 had localized "patches" of sperm detected in areas distant from sperm-negative biopsy sites. In 1 case, a pregnancy was achieved with later biopsy and sperm extraction "directed" by previous FNA. CONCLUSIONS: For sperm detection, testis FNA provides equivalent or better information than a testis biopsy. FNA can localize areas of sperm production within the testis and accurately guide sperm extraction procedures in men with NOA.

KTP-532 laser ablation of urethral strictures.

In 1988 the KTP-532 laser was used to ablate a series of benign urethral strictures. Rather than using a single incision as in urethrotomy, strictures were treated with 360-degree contact photoradiation. Thirty-one male patients, average age 53.2 years, received thirty-seven treatments; 6 patients underwent a second laser treatment. Stricture etiology was commonly iatrogenic (32%), traumatic (16%), and postgonococcal (10%). Stricture location included mainly bulbar (49%), membranous (20%), and penile (12%) areas. The surgical technique consisted of circumferential ablation, followed by Foley catheter placement (mean, 10 days). Follow-up on 29 of 31 patients ranged from one to sixteen months (mean 9.7). Complete success occurred in 17 patients (59%) who had no further symptoms or instrumentation. Partial success was seen in 6 patients (20.5%) with symptom, but not stricture, recurrence. Six patients (20.5%) failed therapy, requiring additional surgery or regular dilations. No complications were seen. Although longer assessment is required, KTP-532 laser ablation of urethral strictures appears efficacious.

The clinical characteristics of 82 patients with Sertoli cell-only testis histology.

OBJECTIVE: To determine the clinical and laboratory features associated with Sertoli cell-only testis histology. DESIGN: Case-controlled retrospective analysis. SETTING: University-based male infertility clinic. PATIENTS: Eighty-two patients with germ cell aplasia histology on testis biopsy from 1984 to 1994. Hormonal findings from a control group of 34 fertile men with normal semen analyses were used for comparison. MAIN OUTCOME MEASURES: History, physical examination, semen analysis, and endocrine findings. RESULTS: Sertoli cell-only histology was observed in 82 of 652 (13%) biopsies performed in infertile men with azoospermia. A major medical illness or genital anomaly (hypospadias, cryptorchidism) was found in 31 men (38%). On physical examination, 75% of patients had small testis (< 18 mL volume) and 45% had abnormally soft testis. On hormone evaluation only serum FSH concentration was elevated significantly (33 mIU/mL [33 IU/L]) compared with fertile controls (7.1 mIU/L [7.1 IU/L]). Patients (6%) with confirmed elevations in serum PRL had lower FSH values than those men with normal PRL levels. Normal ejaculate volumes were observed in all patients. CONCLUSIONS: In approximately 38% of patients, germ cell aplasia is associated with underlying disease states; in the remainder it is largely idiopathic. The physical examination is often characteristic, along with an elevated serum FSH concentration. Normal FSH levels tend to be associated with hyperprolactinemia in these men.

Birth after intracytoplasmic sperm injection with use of testicular sperm from men with Kartagener/immotile cilia syndrome.

OBJECTIVE: To describe two cases of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with testicular sperm in men with immotile cilia syndromes. DESIGN: Case report. SETTING: A university-based male infertility clinic and assisted reproduction unit. PATIENT(S): Two couples with male factor infertility due to Kartagener/immotile cilia syndrome. INTERVENTION(S): IVF/ICSI with testicular sperm. MAIN OUTCOME MEASURE(S): Semen characteristics, sperm viability, fertilization rate, and pregnancy. RESULT(S): With testicular sperm, the two pronuclear fertilization rates were 63% and 60% in two cases. One case resulted in the birth of normal healthy girl. CONCLUSION(S): With testicular sperm, successful oocyte fertilization after ICSI in couples with male Kartagener/immotile cilia syndrome is possible despite the lack of sperm motility.

Does response to treatment of ejaculatory duct obstruction in infertile men vary with pathology?

OBJECTIVE: To describe the pathology-specific response to transurethral resection of ejaculatory ducts (TURED) in patients with complete or partial ejaculatory duct obstruction and to evaluate the role of TURED in light of powerful assisted reproductive technologies. DESIGN: Retrospective clinical study. SETTING: University hospital. PATIENT(S): Thirty-eight infertile men with obstruction of the ejaculatory ducts. INTERVENTION(S): Diagnosis by transrectal ultrasonography or magnetic resonance imaging, and treatment with TURED. MAIN OUTCOME MEASURE(S): Changes in semen variables, pregnancy outcomes, and complication rates were analyzed before and after surgery. RESULT(S): Improvement in semen variables was significantly better in patients with partial obstruction (94%) of ducts than in those with complete obstruction (59%) (P=.04). Cystic obstruction, especially midline and eccentric cysts, responded best to TURED. Before surgery, all patients were candidates for IVF/ICSI; after surgery, 32% of azoospermic men and 81% of oligospermic men conceived spontaneously or were referred for IUI instead of IVF/ICSI. CONCLUSION(S): Ejaculatory duct obstruction due to cysts appears to respond best to TURED. In addition, TURED may decrease the need for IVF/ICSI as primary treatment in many cases. Finally, TURED may allow IVF/ICSI to be performed with ejaculated rather than surgically retrieved sperm.

Testis sperm extraction and intracytoplasmic sperm injection guided by prior fine-needle aspiration mapping in patients with nonobstructive azoospermia.

OBJECTIVE: To evaluate intracytoplasmic sperm injection (ICSI) outcomes in a cohort of men with nonobstructive azoospermia who underwent prior fine-needle aspiration (FNA) "maps" to localize sperm and guide testis sperm extraction (TESE). DESIGN: Retrospective clinical study. SETTING: University-based infertility practice. PATIENT(S): A consecutive cohort of 19 infertile, azoospermic men. INTERVENTION(S): Couples underwent IVF-ET in which TESE procedures were informed and directed by prior FNA maps of the testis. MAIN OUTCOME MEASURE(S): Sperm retrieval and pregnancy rates. RESULT(S): In 21 IVF-ET and ICSI cycles, sufficient sperm for all oocytes were retrieved in 20 TESE attempts (95%). A mean of 3.1 biopsies per patient were required, with an average size of 72 mg. Mean operative time for the TESE procedure was 88 minutes. Overall, the two-pronuclear fertilization rate was 66%; ongoing clinical pregnancies were obtained in 10 of 21 initiated cycles (48%). CONCLUSION(S): In an effort to reduce IVF-ET cancellation rates in cases of nonobstructive azoospermia, diagnostic testis FNA can define those patients who are good candidates for TESE. It also directs sperm retrieval and minimizes tissue removal from nonobstructed testes.

Immunologic infertility.

The existence of immunologic infertility is well grounded in the theories of humoral and cell-mediated immunology. Both of these immune system arms can theoretically and experimentally be shown to play roles in states of male factor infertility. Although much progress has been made in relating immunopathology to infertility, several problems plague the investigation and diagnosis: the use of multiple, different assay systems; the lack of properly controlled experimental work; a need to oversimplify a tremendously complex immunologic network for clinical purposes; and a dearth of communication between basic science and clinical investigators. Unlike the established role of humoral or antibody-mediated infertility, the way cell-mediated immunity induces reproductive failure is just beginning to be understood. Two active areas of investigation include a delineation of active immunosuppression in the normal testicular environment and the pathologic deficits induced by cytokines in cases of leukospermia. Other research seeks to define functional, sperm-specific antigens to which autoantibodies are directed and to elucidate the mechanisms of antibody-mediated infertility at various sites in the reproductive tract. The diagnosis of immunologic infertility still remains one of exclusion. When the diagnosis is made, it is necessary to define precisely which functional reproductive deficits exist, through an analysis of semen parameters, postcoital tests, sperm penetration assays, and tests of acrosomal reactivity. This is essential because the therapeutic options are broad and can involve significant side effects and expense. Proven modalities include sperm manipulation, direct sperm insemination, donor insemination, systemic immunosuppression, and assisted reproductive techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Ejaculatory physiology and dysfunction.

The sequence of events encompassing ejaculation has been well described. Multiple disease processes can result in ejaculatory dysfunction. Evaluation and subsequent treatment of ejaculatory dysfunction is possible using behavioral, mechanical, and medical and surgical modalities. Further elucidation of ejaculation is now taking place at the molecular level.

Fertility issues and their management in men with testis cancer.

Although a curable malignancy, testis cancer and its treatment have unique associated morbidities that largely affect reproductive dysfunction. In this focused review, the factors that contribute to infertility in men with testis cancer are outlined. The treatment-specific risks to fertility that accompany cancer management are also discussed. Contemporary methods of overcoming infertility in testis cancer patients are addressed, and several exciting and promising experimental approaches to the preservation or restoration of fertility for men with testis cancer are presented.

Effects of dietary selenium on sperm motility in healthy men.

A deficiency of dietary selenium leads to immotile, deformed sperm and infertility in rats, whereas supplementation of the diet with selenium compounds has been associated with both beneficial and deleterious effects on sperm function, depending on the chemical form of selenium. We conducted a randomized, controlled, and blinded intervention study on the effects of selenium in food on semen quality. Eleven healthy men were fed a controlled diet of foods naturally high or low in selenium for 120 days while confined in a metabolic research unit. Dietary selenium was 47 microg/d for the first 21 days, then either 13 microg/d or 297 microg/d for 99 days, resulting in significant changes in selenium concentrations in blood and semen. Seminal plasma selenium concentration increased 50% with high selenium and decreased 40% with low selenium. The fraction of motile sperm in the high-selenium group decreased by 32% by week 13 and ended 18% lower than baseline. Selenium concentrations changed in seminal plasma but not in sperm, and serum androgen concentrations were unchanged in both groups, indicating this effect was neither androgen dependent nor caused by a change in the selenium supply to the testes. Serum triiodothyronine decreased and thyroid-stimulating hormone increased in the high-selenium group, suggesting that altered thyroid hormone metabolism may have affected sperm motility. Although this decrease in sperm motility does not necessarily predict decreased fertility, the increasing frequency of selenium supplementation in the healthy population suggests the need for larger studies to more fully assess this potential side effect.

The need for epididymovasostomy at vasectomy reversal plateaus in older vasectomies: a study of 1229 cases.

Vasectomy reversal involves either vasovasostomy (VV) or epididymovasostomy (EV), and rates of epididymal obstruction and EV increase with time after vasectomy. However, as older vasectomies may not require EV for successful reversal, we hypothesized that sperm production falls after vasectomy and can protect the system from epididymal blowout. Our objective was to define how the need for EV at reversal changes with time after vasectomy through a retrospective review of consecutive reversals performed by three surgeons over a 10-year period. Vasovasotomy was performed with Silber score 1-3 vasal fluid. EVs were performed with Silber score 4 (sperm fragments; creamy fluid) or 5 (sperm absence) fluid. Reversal procedure type was correlated with vasectomy and patient age. Post-operative patency rates, total spermatozoa and motile sperm counts in younger (<15 years) and older (>15 years) vasectomies were assessed. Simple descriptive statistics determined outcome relevance. Among 1229 patients, 406 had either unilateral (n = 252) or bilateral EV's (n = 154) constituting 33% (406/1229) of reversals. Mean patient age was 41.4±7 years (range 22-72). Median vasectomy interval was 10 years (range 1-38). Overall sperm patency rate after reversal was 84%. The rate of unilateral (EV/VV) or bilateral EV increased linearly in vasectomy intervals of 1-22 years at 3% per year, but plateaued at 72% in vasectomy intervals of 24-38 years. Sperm counts were maintained with increasing time after vasectomy, but motile sperm counts decreased significantly (p < 0.001). Pregnancy, secondary azoospermia, varicocoele and sperm granuloma were not assessed. In conclusion, and contrary to conventional thinking, the need for EV at reversal increases with time after vasectomy, but this relationship is not linear. EV rates plateau 22 years after vasectomy, suggesting that protective mechanisms ameliorate epididymal 'blowout'. Upon reversal, sperm output is maintained with time after vasectomy, but motile sperm counts decrease linearly, suggesting epididymal dysfunction influences semen quality after reversal.

A stable isotope-mass spectrometric method for measuring human spermatogenesis kinetics in vivo.

PURPOSE: Currently it is thought to take 60 to 70 days to produce and ejaculate human sperm. This estimate is derived mainly from a single older, descriptive, kinetic analysis of spermatogenesis. We developed a noninvasive method to assess germ cell turnover time accurately in vivo using stable isotope labeling and gas chromatography/mass spectrometry analyses. We confirmed the postulated length of a normal cycle of spermatogenesis. MATERIALS AND METHODS: A total of 11 men with normal sperm concentrations ingested (2)H(2)O daily for 3 weeks. Semen samples were collected every 2 weeks for up to 90 days. Label incorporation into sperm DNA was quantified by gas chromatography/mass spectrometry, allowing calculation of the percent of new cells present. A cycle of sperm production was determined as the lag time until labeled sperm appeared in the ejaculate. RESULTS: Labeled sperm were detected after a mean +/- SD of 64 +/- 8 days (range 42 to 76). In 1 subject the time lag was 42 days but it was at least 60 in all other subjects. In most subjects plateau labeling in sperm was not attained. In 2 subjects the rise and fall of the labeling curve was steep and reached greater than 85% new cells, suggesting rapid washout of old sperm in the epididymal reservoir. CONCLUSIONS: This direct kinetic assessment confirms a course of spermatogenesis that is on the shorter side of traditional estimates based on prior analyses. In addition, the variability observed in healthy men suggests that characteristics such as the epididymal reservoir effect may influence the modeling of in vivo spermatogenesis.

Temporal progression of recombination in human males.

To date, immunocytology has been used in humans to detect a limited number of meiotic proteins: components of the synaptonemal complex (SCP1 and SCP3) and some proteins known to participate in recombination events, such as MLH1 or RAD51. However, the colocalization or coexistence of proteins known to participate during the different stages of human meiosis remains largely unstudied, and these studies could provide important clues about the mechanics of recombination. This work reports the relative timing and localization of five different meiotic proteins that have previously been implicated in human homologous recombination [RAD51, replication protein A (RPA), MSH4, MLH1 and MLH3]. MSH4 foci appear concurrently with synapsis initiation at zygotene, shortly after the first RAD51 foci are detected. The presence of RPA in MSH4 foci was noted, suggesting that these two proteins may act co-operatively. Both RPA and MSH4 foci reach maximal numbers at the end of zygotene, when synapsis is concluding. From this point, RPA foci all but disappear by the end of pachytene, whereas MSH4 foci decline to a stable number at mid-pachytene, where they localize with MLH1/MLH3 recombination sites. We discuss a possible role for MSH4 in synapsis initiation and/or maintenance.

Meiotic studies in an azoospermic human translocation (Y;1) carrier.

A reciprocal translocation between the long arm of the Y chromosome and the long arm of chromosome 1 was observed in an infertile man with non-obstructive azoospermia. The study was performed using a combination of techniques: immunocytogenetic analysis, which allows the detection of synaptonemal complexes (SCs) and recombination sites (MLH1) simultaneously, and fluorescence in-situ hybridization analysis. Meiotic pairing analysis on 100 pachytene spreads showed the presence of a quadrivalent containing chromosomes 1 and Y. There were many abnormalities in chromosome pairing and recombination. These abnormalities included a great reduction of recombination events (as many as one fifth of the SCs had no MLH1 foci), and high proportions of unpaired regions and discontinuities in the SCs. We discuss the possibility that infertility in this patient may be due to transcriptional repression of part of chromosome 1 involved in the translocation, silencing some genes necessary for the progression of meiosis and causing defective meiotic pairing and recombination.

Hypo-osmotic swelling can accurately assess the viability of nonmotile sperm.

Viable, healthy sperm are preferred for oocyte fertilization with intracytoplasmic sperm injection. Currently, motility is the most widely applied measure of sperm viability. However, with this criterion, viable but immotile sperm are overlooked as candidates for micromanipulation. Supravital stains identify viable sperm but may be toxic to the gamete or embryo. We tested the hypothesis that hypo-osmotic swelling, developed to assess sperm membrane integrity, can accurately determine sperm viability. Thawed sperm from 12 fertile donors were exposed to a hypo-osmotic solution and to two supravital stains. A total of 2,010 sperm were assessed for tail coiling after a 30-min exposure to hypoosmotic solution. By supravital stains, 31% of thawed sperm were viable; 23% showed tail coiling. Among coiled-tail sperm, 100% were viable by supravital stain. As a measure of viability, tail coiling exhibited a sensitivity of 30%, specificity of 100%, and positive predictive value of 100% compared to supravital stains. With a 60-min hypo-osmotic incubation, the specificity (89%) and positive predictive value (78%) decreased significantly (P < 0.05). Therefore, hypo-osmotic swelling accurately detects viable sperm among a nonmotile population. Assay accuracy, however, is very sensitive to the incubation time in hypo-osmotic solution.